Lysate composition and application thereof in preparation of reagent for extracting genome DNA

Abstract

The invention belongs to the technical field of molecular biology and in-vitro diagnosis, and particularly discloses a lysis solution composition and application thereof in preparation of a reagent for extracting genome DNA. The lysate composition comprises the following components: a pH regulator, 0.05-1.0% (v / v) of a nonionic surfactant, 0.1-2 mg / mL of bovine serum albumin, 0.5-5 mM of a reducing agent and 50-500 g / mL of protease K, and the pH value of the lysate composition is 7.0-8.0. The invention provides the genome DNA quick release lysate which is reasonable in composition and simple and convenient to operate, efficient release and stable storage of DNA in a complex biological sample are realized under the condition that a traditional purification step is not needed, and the obtained DNA can be directly used for nucleic acid amplification detection such as PCR (Polymerase Chain Reaction) and LAMP (Loop-Mediated Isothermal Amplification), so that the detection efficiency, repeatability and application convenience are improved; the actual requirements of rapid molecular diagnosis and on-site detection are met.

Description

Technical Field

[0001] This application relates to the fields of molecular biology and in vitro diagnostics, and more particularly to a lysis buffer composition and its application in the preparation of reagents for extracting genomic DNA. Background Technology

[0002] Nucleic acid detection technology is a core tool in molecular diagnostics, biomedical research, and genetic analysis. The efficient release and stable acquisition of genomic DNA (gDNA) are prerequisites for accurate subsequent nucleic acid amplification, genotyping, and molecular detection. Currently, for complex biological samples such as blood and swabs, existing technologies commonly employ silica gel membrane column methods, magnetic bead methods, or organic solvent extraction methods for DNA extraction. These methods typically rely on multi-step procedures, including sample lysis, protein removal, nucleic acid adsorption, washing, and elution. These processes are not only cumbersome and time-consuming but also require high levels of experimental conditions and operator skill, hindering the widespread application of rapid detection and point-of-care testing (POCT). Furthermore, frequent centrifugation or magnetic separation steps limit the use of these methods in primary healthcare institutions and resource-constrained environments.

[0003] To simplify the DNA release process, some studies have attempted to use chemical lysis systems to directly release DNA from whole blood or cell samples. For example, some existing technologies utilize a Tris-HCl buffer system to maintain a stable solution pH, thereby reducing DNA hydrolysis; add proteinase K to lyse cell structures and degrade nucleic acid-binding proteins, thus promoting DNA release; and simultaneously use nonionic surfactants (such as Tween-20) to disrupt cell membrane and nuclear membrane structures, improving lysis efficiency.

[0004] However, the simplified lysis system described above still has certain shortcomings in practical applications. On the one hand, blood samples are rich in hemoglobin, immunoglobulins, and other inhibitory components, which can easily inhibit downstream nucleic acid amplification reactions. On the other hand, during lysis and preservation, protease activity is easily affected by the oxidative environment, thus affecting DNA release efficiency and reproducibility. In addition, some existing lysis systems still struggle to balance DNA stability, inhibitor tolerance, and downstream amplification compatibility, making it difficult to meet the needs of rapid, direct amplification detection.

[0005] Therefore, there is an urgent need to provide a nucleic acid release system that is rationally composed, easy to operate, and capable of efficiently releasing and stably preserving DNA in complex biological samples, in order to overcome the problems of complex operation, significant amplification inhibition, and insufficient detection consistency in existing technologies. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this application is to provide a lysis buffer composition and its application in the preparation of reagents for extracting genomic DNA, so as to solve the problems of cumbersome, time-consuming and easily introduced amplification inhibition in the DNA extraction process of complex biological samples such as whole blood in the prior art.

[0007] To achieve the above and other related objectives, this application provides a lysis buffer composition comprising the following components: a pH adjuster, a nonionic surfactant, bovine serum albumin (BSA), a reducing agent, and proteinase K. The nonionic surfactant has a volume concentration of 0.05–1.0% (v / v), the bovine serum albumin has a concentration of 0.1–2 mg / mL, the reducing agent has a concentration of 0.5–5 mM, and the proteinase K has a concentration of 50–500 µg / mL. The reducing agent is used to prevent oxidative inactivation of proteinase K during high-temperature lysis and to maintain the proteolytic ability of proteinase K. The pH of the lysis buffer composition is 7.0–8.0.

[0008] Furthermore, the pyrolysis solution composition is a water-soluble composite pyrolysis system.

[0009] Furthermore, the pH adjuster is selected from buffer solutions with a pH of 7.0 to 8.0.

[0010] Furthermore, the buffer solution is selected from Tris-HCl buffer solution.

[0011] Furthermore, the nonionic surfactant is selected from the Tween series of nonionic surfactants, including but not limited to a combination of one or more of Tween-20, Tween-40, and Tween-60.

[0012] Furthermore, the reducing agent is selected from dithiothreitol (DTT).

[0013] Furthermore, the lysis buffer composition comprises the following components: a) Tris-HCl buffer, concentration 10~100 mM, pH 7.0~8.0; b) Tween-20, with a volume concentration of 0.05~1.0% (v / v); c) Bovine serum albumin, at a concentration of 0.1–2 mg / mL; d) Dithiothreitol, concentration 0.5~5 mM; e) Proteinase K, at a concentration of 50~500 µg / mL.

[0014] Furthermore, the lysis buffer composition comprises the following components: a) Tris-HCl buffer, concentration 50 mM, pH 7.0~8.0; b) Tween-20, volume concentration of 1.0% (v / v); c) Bovine serum albumin, at a concentration of 1 mg / mL; d) Dithiothreitol, concentration 5 mM; e) Proteinase K, at a concentration of 200 µg / mL.

[0015] This application also provides the use of the lysis buffer composition described above in the preparation of reagents for extracting genomic DNA.

[0016] Furthermore, the reagent releases and preserves the genomic DNA of the biological sample, which includes, but is not limited to, whole blood samples.

[0017] Furthermore, the genomic DNA extracted by the reagent is used for nucleic acid amplification and detection.

[0018] As described above, the lysis buffer composition of this application and its application in the preparation of reagents for extracting genomic DNA have the following beneficial effects: This application aims to address the problems of cumbersome, time-consuming, and easily amplification-inhibiting DNA extraction processes in complex biological samples such as whole blood. It provides a rationally composed and easy-to-use rapid release and lysis buffer for genomic DNA, achieving efficient release and stable preservation of DNA from complex biological samples such as whole blood without the need for traditional purification steps. This allows the obtained DNA to be directly used for nucleic acid amplification detection such as PCR and LAMP, thereby improving detection efficiency, repeatability, and ease of application, and meeting the practical needs of rapid molecular diagnostics and on-site testing.

[0019] Compared with existing technologies, this application achieves rapid and efficient release of genomic DNA from complex biological samples such as whole blood through a synergistic lysis system composed of a pH adjuster (such as Tris-HCl), a nonionic surfactant (such as Tween-20), proteinase K, a reducing agent (such as DTT), and BSA. DNA that can be directly used for downstream detection can be obtained by heating at 90-100 °C for 8-12 min. The supernatant does not need to undergo column purification or magnetic bead extraction, which significantly simplifies the sample pretreatment process and shortens the detection time. Among them, the pH adjuster maintains a stable reaction pH environment to protect DNA integrity, the nonionic surfactant reduces the inhibition of polymerase reaction while ensuring lysis efficiency, proteinase K continuously and efficiently degrades proteins and nucleases under the protection of the reducing agent, and BSA effectively buffers inhibitory components such as hemoglobin in blood, thereby significantly improving the success rate and reproducibility of subsequent PCR or sequencing reactions. Attached Figure Description

[0020] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application. It is obvious that the drawings described below are merely some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0021] In the attached diagram: Figure 1 The effect of different concentrations of Tween-20, BSA, DTT, and proteinase K on PCR amplification efficiency is shown in Example 1 of this application.

[0022] Figure 2 The PCR amplification curves are typical CC, TT, and TC type samples from Example 1 of this application.

[0023] Figure 3 The results are Sanger sequencing results of typical CC, TT, and TC type samples in Example 1 of this application. Detailed Implementation

[0024] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0025] In this application, unless otherwise stated, the term "multiple" means two or more.

[0026] The character " / " indicates that the objects before and after it are in an "or" relationship. For example, A / B means: A or B.

[0027] The term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0028] One embodiment of this application provides a lysis buffer composition comprising the following components: a pH adjuster, a nonionic surfactant, BSA, a reducing agent, and proteinase K, wherein the volume concentration of the nonionic surfactant is 0.05-1.0% (v / v), the concentration of BSA is 0.1-2 mg / mL, the concentration of the reducing agent is 0.5-5 mM, and the concentration of proteinase K is 50-500 µg / mL; the reducing agent is used to prevent oxidative inactivation of proteinase K during high-temperature lysis and to maintain the proteolytic ability of proteinase K; the pH of the lysis buffer composition is 7.0-8.0.

[0029] In some embodiments of this application, the pyrolysis composition is a water-soluble composite pyrolysis system.

[0030] In some embodiments of this application, the pH adjuster is selected from a buffer solution with a pH of 7.0 to 8.0, and the buffer solution is preferably a Tris-HCl buffer solution.

[0031] In some embodiments of this application, the nonionic surfactant is selected from the Tween series of nonionic surfactants, including but not limited to a combination of one or more of Tween-20, Tween-40 and Tween-60.

[0032] In some embodiments of this application, the reducing agent is selected from DTT.

[0033] In some embodiments of this application, the lysis buffer composition comprises the following components: a) Tris-HCl buffer, concentration 10~100 mM, pH 7.0~8.0; b) Tween-20, with a volume concentration of 0.05~1.0% (v / v); c) Bovine serum albumin, at a concentration of 0.1–2 mg / mL; d) Dithiothreitol, concentration 0.5~5 mM; e) Proteinase K, at a concentration of 50~500 µg / mL.

[0034] In an exemplary embodiment of this application, the lysis buffer composition comprises the following components: a) Tris-HCl buffer, 50 mM, pH 7.0–8.0; b) Tween-20, volume concentration of 1.0% (v / v); c) Bovine serum albumin, at a concentration of 1 mg / mL; d) Dithiothreitol, concentration 5 mM; e) Proteinase K, at a concentration of 200 µg / mL.

[0035] Another embodiment of this application provides the use of the lysis buffer composition described above in the preparation of reagents for extracting genomic DNA.

[0036] In some embodiments of this application, the reagent releases and preserves the genomic DNA of a biological sample, including but not limited to whole blood samples.

[0037] In some embodiments of this application, the genomic DNA extracted by the reagent is used for nucleic acid amplification detection, such as single nucleotide polymorphism (SNP) detection in whole blood samples.

[0038] The embodiments described above provide a lysis buffer composition for the rapid release of gDNA from complex biological samples, which is particularly suitable for the direct release of DNA from whole blood samples and is directly compatible with downstream nucleic acid amplification reactions (such as PCR and sequencing).

[0039] The mechanisms of action of each component are as follows: a) pH adjustment, such as Tris-HCl buffer: used to maintain the pH stability of the solution during the lysis reaction, inhibit acidic or alkaline degradation of DNA, and improve the structural integrity of DNA; b) Nonionic surfactants, such as Tween-20, enhance cell lysis by disrupting the lipid structure of cell and nuclear membranes, while avoiding the significant inhibition of downstream amplification reactions by strong detergents. c) BSA: As a protein stabilizer and inhibitor buffer, it can effectively adsorb hemoglobin, peptides and other inhibitory components in blood samples, and improve the tolerance of downstream nucleic acid amplification systems; d) Reducing agents, such as DTT: Reducing agents are used to prevent proteinase K from being oxidized and deactivated during high-temperature cleavage, thereby maintaining its continuous proteolytic ability; e) Proteinase K: Used to efficiently degrade cellular proteins, nucleic acid-binding proteins and endogenous nucleases, promoting the full release of gDNA and preventing its degradation.

[0040] The above components work synergistically in the same lysis system, ensuring both DNA release efficiency, stability, and amplification compatibility.

[0041] This application can be widely used in clinical molecular diagnostics, genetic disease and SNP detection, nucleic acid detection of infectious disease pathogens, biomarker analysis, and rapid detection and POCT scenarios combined with nucleic acid detection technologies such as PCR and LAMP isothermal amplification.

[0042] The following specific examples illustrate this application in detail. It should also be understood that the following examples are only for illustrative purposes and should not be construed as limiting the scope of protection of this application. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of this application fall within the scope of protection of this application. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0043] Example 1 I. Preparation of 5 mL lysis buffer system Add the components in the order specified in Table 1, mix well and set aside: Table 1 II. Methods for extracting gDNA from whole blood The DNA release from whole blood samples was performed using the above-mentioned lysis buffer, and the specific steps are as follows: a) Take 50 µL of whole blood sample into a centrifuge tube; b) Add 50 µL of lysis buffer and mix well; c) Heat the mixture at 100℃ for 10 min to complete cell lysis and protein degradation; d) After heating, centrifuge at high speed for 10 min; e) The supernatant can be used as a gDNA solution and can be directly used for subsequent nucleic acid amplification and detection.

[0044] III. Evaluation of Blood gDNA Extraction Efficacy The extraction efficiency of blood gDNA was evaluated using the C677T SNP site as the detection target. The total volume of the PCR reaction system was 20 µL, including 10 µL of 2× PCR Mix, 200 nM each of upstream and downstream primers, 1× Eva Green dye, 1 µL of extracted gDNA template, and the remaining volume was made up to 20 µL with nuclease-free water. The amplification program was set as follows: 95 °C pre-denaturation for 5 min; followed by 45 cycles, each cycle including 95 °C denaturation for 15 s, 60 °C annealing / extension for 30 s, with fluorescence signals acquired in real time during the extension / extension phases. The extraction efficiency of blood gDNA was comprehensively evaluated by the amplification curve morphology and fluorescence signal intensity. In addition, the Sanger sequencing reaction system was consistent with the above PCR reaction system, except that Eva Green dye was not added. The obtained amplification products were used for subsequent sequencing analysis to further verify the quality and accuracy of the extracted gDNA. Upstream primer sequence: CCCTCACCTGGATGGGAA, downstream primer sequence: TCTTCATCCCTCGCCTTGA.

[0045] like Figure 1 As shown, by changing the volume concentration of Tween-20 in the lysis buffer (0.25%, 0.5%, 1%, 2%, 4%), the concentration of BSA (0.25, 0.5, 1, 2, 4 mg / mL), the concentration of DTT (1, 2, 5, 10, 20 mM), and the concentration of proteinase K (50, 100, 200, 400, 600 μg / mL), it was found that as the concentrations of Tween-20, BSA, DTT, and proteinase K increased, the PCR amplification efficiency first increased and then decreased. The highest PCR amplification efficiency was achieved when the concentration of Tween-20 was 1%, the concentration of BSA was 1 mg / mL, the concentration of DTT was 5 mM, and the concentration of proteinase K was 200 μg / mL. Figure 2 As shown, the PCR amplification results indicated that the CC, TT, and TC genotypes all exhibited typical S-shaped amplification curves with stable fluorescence signal growth, while the negative control (NTC) showed no amplification signal, indicating that the reaction system was free of non-specific amplification and contamination. Figure 3 As shown, Sanger sequencing results further confirmed that the CC, TT, and TC genotypes were accurately and reliably identified. The above experimental results demonstrate that whole blood samples processed using the lysis system described in this application can obtain stable and clear amplification signals. The released DNA is of good quality and high stability, and exhibits good compatibility with downstream amplification and sequencing analyses. This fully demonstrates that this method has significant technical advantages in terms of lysis efficiency, detection reliability, and ease of operation, making it suitable for rapid molecular detection and POCT applications, and possessing good technical feasibility and economic value for widespread application.

[0046] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A pyrolysis solution composition, characterized in that: The lysis buffer composition comprises the following components: pH adjuster, nonionic surfactant, bovine serum albumin, reducing agent and proteinase K, wherein the volume concentration of the nonionic surfactant is 0.05-1.0%, the concentration of the bovine serum albumin is 0.1-2 mg / mL, the concentration of the reducing agent is 0.5-5 mM, and the concentration of the proteinase K is 50-500 µg / mL; The reducing agent is used to prevent proteinase K from being oxidized and deactivated during high-temperature cleavage, and to maintain the proteolytic ability of proteinase K. The pH of the lysis buffer composition is 7.0 to 8.

0.

2. The pyrolysis fluid composition according to claim 1, characterized in that: The pyrolysis solution composition is a water-soluble composite pyrolysis system.

3. The pyrolysis solution composition according to claim 2, characterized in that: The pH adjuster is selected from buffer solutions with a pH of 7.0 to 8.

0.

4. The pyrolysis fluid composition according to claim 3, characterized in that: The buffer solution is selected from Tris-HCl buffer.

5. The pyrolysis solution composition according to claim 1, characterized in that: The nonionic surfactant is selected from the Tween series of nonionic surfactants; And / or, the reducing agent is selected from dithiothreitol.

6. The pyrolysis fluid composition according to claim 5, characterized in that: The nonionic surfactant is selected from at least one of Tween-20, Tween-40 and Tween-60.

7. The pyrolysis solution composition according to any one of claims 1 to 6, characterized in that: The lysis buffer composition comprises the following components: a) Tris-HCl buffer, concentration 10~100 mM, pH 7.0~8.0; b) Tween-20, volume concentration of 0.05~1.0%; c) Bovine serum albumin, at a concentration of 0.1–2 mg / mL; d) Dithiothreitol, concentration 0.5~5 mM; e) Proteinase K, at a concentration of 50~500 µg / mL.

8. The pyrolysis composition according to claim 7, characterized in that: The lysis buffer composition comprises the following components: a) Tris-HCl buffer, concentration 50 mM, pH 7.0~8.0; b) Tween-20, volume concentration 1.0%; c) Bovine serum albumin, at a concentration of 1 mg / mL; d) Dithiothreitol, concentration 5 mM; e) Proteinase K, at a concentration of 200 µg / mL.

9. Use of the lysis buffer composition according to any one of claims 1 to 8 in the preparation of reagents for extracting genomic DNA.

10. The application according to claim 9, characterized in that: The reagent releases and preserves the genomic DNA of the biological sample; And / or, the genomic DNA extracted by the reagent is used for nucleic acid amplification detection.

Images